Phytosulfokine (PSK) is a tyrosine-sulfated peptide that is widely distributed in plants, participating in cell proliferation, differentiation and innate immunity. The potential role of PSKs in nodulation in legumes has not been reported. In this work, five PSK precursor genes were identified in Lotus japonicas, designated as LjPSK1-5. Three of them (LjPSK1, LjPSK4 and LjPSK5) were found to be expressed in nitrogen-fixing root nodules. LjPSK1 and LjPSK4 were not induced at the early stage of nodulation. Interestingly, while the expression of LjPSK4 was also found in spontaneous nodules without rhizobial colonization, LjPSK1 was not induced in these pseudo nodules. Promoter-GUS analysis revealed that LjPSK1 was highly expressed in enlarged symbiotic cells of nodules. Exogenous addition of 1 μM synthetic PSK peptide resulted in increased nodule numbers per plant. Consistently, the number of mature nodules, but not the events of rhizobial infection and nodule initiation, was increased by overexpressing LjPSK1 in transgenic hairy roots, where the expression of jasmonate responsive genes was found to be repressed. These results suggest that PSK is a new peptide signal that regulates nodulation in legumes, probably through cross-talking with other phytohormones.